Recent advances in sequencing technologies have led to the discovery of a multitude of novel zoonotic viruses, some of which are closely related to known human pathogens. In most cases, only partial viral genome sequences were identified, and no infectious viruses were isolated, hampering research aimed at assessing the pathogenic potential of these novel viruses. A prime example of a sequence-based novel virus is Lloviu virus (LLOV), the newest member of the filovirus family. LLOV is closely related to the highly pathogenic ebola- and marburgviruses that cause severe disease in humans with extraordinarily high case fatality rates. The genomic viral RNA of LLOV was isolated from dead bats in Spain and is rudimentary. Important sequence elements located at the LLOV genome ends are missing. These include the antigenomic promoter that is essential for viral genome replication. Therefore, studies addressing LLOV genome replication and transcription, or pathogenesis have been impossible to date. In this proposal, we bring together expertise in the field of filoviral replication and transcription, rescue of filovirus clones, filovirus pathogenesis, and statistical modeling to generate infectious LLOV clones for in vitro and in vivo studies. By complementing the missing LLOV sequence with homologous sequences from related filoviruses, we have successfully established a hybrid LLOV minigenome system, enabling studies of LLOV replication and transcription for the first time. Based on this system, we will utilize LLOV minigenome systems in combination with statistical modeling to optimize the complemented promoter sequences (Aim 1). These optimized minigenome systems build the foundation for the generation and rescue of infectious full-length LLOV clones. The generated LLOV clones will then be characterized in in vitro studies. We will determine replication kinetics and host response signatures in primary human cells (Aim 2). Finally, we will perform pathogenesis studies with LLOV using a small animal model of filovirus infection (Aim 3). These studies will be instrumental to get a better understanding of the virulence of LLOV and its potential to cause disease in animals and humans. The platform developed in this project to assess the pathogenic potential of novel viruses can be adapted to other nonsegmented negative-sense RNA viruses and would therefore be of great benefit to the scientific community.